Principles of Biacore measurements. Biacore™ systems from GE Healthcare exploit the phenomenon of surface plasmon resonance (SPR) to monitor the. Biacore AB reserves the right to make changes to the design and content of the Concentration Analysis Handbook without prior notice. Biacore is a registered. Biacore Assay Handbook Edition AA. 1. 1 Introduction discussed in a separate Handbook (Biacore Concentration Analysis Handbook).
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Concentration. ± nM . for calibration-free concentration analysis ( CFCA) does not require a . Biacore Sensor Surface Handbook, version AA. Biacore X Handbook BR Edition AB. 3 and (with the optional Biacore X Plus Package) concentration analyses. GE Healthcare Biacore Concentration Analysis Handbook Contents 1 Introduction Principles of Biacore Biacore in concentration measurement Why use.
Below is how we matched the DMSO concentration of our samples. Regeneration condition For analytes with slow dissociation when the sensorgram does not go back to the background level at the end of the dissociation step , a regeneration step must be performed after each binding assay.
Determine an optimum regeneration condition for each analyte. The regeneration should not change the activity of the immobilized ligand that is, the responses obtained from the binding assays before and after regeneration should be the same.
Binding assay Perform the binding assay was with 1x RB-b. Below is an example of a binding assay. A sensorgram of protein-peptide interaction is shown in Figure 3 and also in the reference Eguchi et al.
Figure 3. Binding assay Recipes The buffers used for immobilization and binding must be optimized for every ligand and analyte. The following recipe worked with our protein and peptide samples.
The sensorgrams were double referenced against the buffer reference signal and the empty sensors by the Data Analysis software 7. The double referenced signals of each association and dissociation phase were combined and exported into a BiaEvaluation 4. The sensorgrams were fitted with the BiaEvaluation software 4.
Five sensors recorded the kinetic titration series, whereas one sensor recorded the buffer reference signal and six sensors were used as sensor reference. Results Ligand immobilization In order to compare the practicality and efficiency between multi cycle kinetics and kinetic titration series using BLI, we used the well-studied interaction of GB1 with IgG.
ScFvs show in comparison to IgGs no avidity.
GB1 was immobilized onto AR2G sensors via amine coupling. We found that at the end of the multiple immobilization procedures the amount of immobilized protein differed for each sensor Tab.
The mean and standard deviation was 1. Thus, homogeneous immobilization of protein to the surface was not possible, because the on-rates of the sensors seem to deviate from each other. The sensors with 1.
However, with declining amount of ligand, the ratio of standard deviation to immobilized ligand grew see Tab. Binding kinetics with parallel sensor kinetics Parallel sensor kinetics is thought to be more precise than a kinetic titration series, because no secondary processes like dissociation of previously bound analyte from the surface impair the measurements.
However, other effects like inhomogeneous coating and differences in sensitivity of single sensors are expected to compromise these precision advantages.
To obtain data from parallel sensor kinetics, five sensor pairs were applied to record the signals received from applying five different analyte concentrations.Inhibition assay with the analyte or analogue attached to the surface and detecting molecule in solution.
Improved red blood cell preservation correlates with decreased loss of bands 3, 41, acetylcholinestrase, and lipids in microvesicles.
This curve is displayed directly on the computer screen during the course of an analysis. Binding affinities can be obtained from the ratio of rate constants, yielding a straightforward characterization of protein-protein interaction. It represents the averaged, squared residual per data point.
Evaluation of concentration measurements in Biacore offers a fully defined four-parameter equation for fitting a curve to the calibration data points.
There is no upper limit for assay procedures that do not impose restrictions on dilution factors involved in sample preparation: in some cases, however, sample preparation procedures may define a maximum permitted dilution, so that the assay will have an upper limit of quantitation.
Among the question that can be addressed with Biacore are: The specificity of biomolecular interactions, investigated by testing the extent of binding between different pairs of molecules.
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